Antibodies recognizing this target with high avidity are substrate for generation of ADC, BiTE and CAR T cells. -turns and a -hairpin stabilized by 2 hydrogen bonds. The VH-VLinterface within the 4H11-scFv is stabilized through an intricate network of 11 hydrogen bonds and a cation- interaction. == Conclusions == Together, our studies offer insight into antibody-MUC16 ectodomain interaction and advance our ability to design agents with potentially improved therapeutic properties over anti-CA125 moiety antibodies. == Supplementary Information == The online version contains supplementary material available at 10.1186/s13048-024-01373-9. Keywords:MUC16, CA125, Ovarian cancer, Antibody drug conjugate, Bispecific T-cell engager, Chimeric antigen receptor-T cells, Crystal structure == Background == The tethered mucin MUC16 protein is physiologically present and intricately controlled in reproductive, respiratory and corneal tissue to protect the epithelium by forming a biological mucosal barrier at the apical surface against hostile environmental conditions and pathogenic infections [14]. However, in several malignancies including ovarian, breast, lung, and pancreatic cancers, it has been reported that overexpression of MUC16 can promote unfavorable characteristics of cancer cells, Rabbit Polyclonal to PARP (Cleaved-Asp214) including changes in cell-to-cell communication, enhanced proliferation, increased accumulation of cancer cells in the G2/M phase with apoptosis resistance, and tumor metastasis. MUC16 overexpression has also been shown to facilitate tumor immune escape via direct suppression of natural killer (NK) and macrophages [49]. MUC16 is composed of 3 major domains and the following subdomains; a heavily glycosylated extracellular region including an N-terminal portion, a tandem-repeated domain interspersed with sea urchin Sperm, Enterokinase, and Agrin (SEA) domain, and a carboxyl-terminal domain. The tandem repeat region encodes the CA125 antigen, a complex, O-glycosylation enhanced epitope that is the cognate receptor for the mesothelin protein [1013]. The carboxyterminal sequence can be further divided into three subdomains composed of an extracellular 61 amino acid juxtamembrane (ectodomain) portion, a transmembrane (TM) region, a 31 amino acid cytoplasmic-tail domain harboring potential phosphorylation sites, and an ezrin binding domain [1416]. The limited tissue expression of MUC16 has made it an attractive candidate for antibody-based, targeted therapy development in high grade LOXL2-IN-1 HCl serous ovarian cancer (HGSOC) [1721]. However, much of this activity has utilized antibodies targeting the tandem repeat region. This strategy has two significant shortcomings: (a) the tandem repeat protein is present in the circulation, acting as an antigen sink and (b) the shed CA125 region promotes off-target effects particularly on mesothelin-expressing LOXL2-IN-1 HCl surfaces [18,19]. Prior therapeutics like abagovomab, oregovomab, and DMUC (Genentech), have targeted the tandem repeat portion of MUC16 [14,19,22]. The OC125/M11 epitopes present in the tandem repeat region are dependent on folding and enhanced by glycosylation processes which have limited potential for antibody targeting of MUC16 [23]. Our prior work suggests that targeting non-CA125 protein epitopes in the proximal MUC16 ectodomain (MUC16ecto) using a murine antibody (m4H11) may still block MUC16 related oncogenic functions [2426]. The sequences of the membrane-proximal MUC16 SEA domains are more divergent than those of distal SEA domains, and because mAb 4H11s epitope is only partially conserved in other SEA domains it is assumed that soluble CA125 would not act as a sink. To mitigate the issues surrounding the use of murine antibodies as human drugs, we set out to engineer and characterize a humanized antibody version against MUC16ecto. With this statement, we evaluated matrigel invasion, antibody drug conjugate (ADC) killing, and Chimeric Antigen Receptor (CAR)-T cell therapy. Finally, we cautiously explored the structural relationships between the MUC16 ectodomain and a humanized version of 4H11 antibodies against MUC16ectotargets. We describe the crystal constructions of a single chain h4H11-scFv and describe its connection with MUC16ectocomposed of 26 residues (31thLQNFTLDRSSVLVDGYSPNRNEPLTG6th; numbering from TM) to understand the binding mechanism through the assessment of the apo and the MUC16ecto-bound scFv at 2.36 and 2.47 resolutions, respectively. Our results reveal conformational variations in the complementarity-determining areas (CDRs) of the free and MUC16-bound 4H11 scFv. This information can be utilized to improve the restorative potential of antibody-based therapies. == Results == == Humanization of 4H11 antibody == To form the basis for clinical development, the 4H11 murine antibody (m4H11) was first modified to provide a CAR for clinical software [20]. This m4H11 antibody experienced the best binding characteristics from our unique antibody marketing campaign and was used LOXL2-IN-1 HCl as the basis of the humanized development [24]. In collaboration with Eureka Therapeutics Inc (Emeryville, CA), the sequence of the m4H11 antibody was used to search international IMmunoGeneTics (IMGT) database for the closest human being antibody platform, and.